Production of cephalosporin C

ABSTRACT

Cephalosporin C is produced in remarkably increased yields by fermentation process using a cephalosporin C-producing polyploid mold belonging to the genus Cephalosporium.

Unite States Patent [191 Kanzaki et al.

[ 1 Dec. 30, 1975 1 PRODUCTION OF CEPHALOSPORIN C [75] Inventors: Toshihiko Kanzaki, l-lyogo;

Tadazumi Fukita, Osaka; Yoichiro Kono, Kyoto, all of Japan [73] Assignee: Takeda Chemical Industries, Ltd.,

Osaka, Japan [22] Filed: Sept. 30, 1974 [21] Appl. No.: 510,861

[56] References Cited UNITED STATES PATENTS 3,776,815 12/1973 Treichler et al. 195/36 R 3,816,257 6/1974 Yamano et al 195/36 R Primary ExaminerAlvin E. Tanenholtz Attorney, Agent, or FirmWenderoth, Lind & Ponack [5 7] ABSTRACT Cephalosporin C is produced in remarkably increased yields by fermentation process using a cephalosporin C-producing polyploid mold belonging to the genus Cephalosporium.

3 Claims, No Drawings PRODUCTION OF CEPHALOSPORIN C This invention relates to the production of cephalosporin-C (hereinafter abbreviated as CPC) by fermentation processes, and is particularly concerned with processes for the production of CPC using a CPC- producing polyploid mold belonging to the genus Cephalosporium.

Methods by which increased yields of CPC produced by fermentation processes may be obtained have been investigated, but few methods are known in which selective breeding of microbial strains is involved. All that is known at the present time are the methods enlisting the help of a strain whose metabolism and biosynthesis of organosulfer compounds have been blocked (Japanese patent application laid open No.1828/197 1) and those in which the CPC-producing ability of microorganisms has been somehow enhanced (U.S. Pat. No. 3,082,155 and Japanese patent application laid open No.26986/1973). Moreover, in regard of the abilities of microorganisms to produce CPC, the fermentation yields are 760 Lg/ml at best in the case of the Japanese patent application laid open No.1828/l971, about 500 ug/ml in the case of US. Pat. No. 3,082,155 and, even in the most satisfactory of all the cases heretofore reported, Japanese patent application laid open No. 26986/1973, the attainable potency is not more than 5,000 ug/ml. I

The present inventors have attempted to find a way to produce CPC in an increased yield through intensive research directed to a genetic improvement of micoorganisms, and have ultimately found that molds possessing excellent CPC-producing abilities can be obtained at a higher frequency from among polyploid molds induced by polyploidizing treatment of CPC-producing microorganisms belonging to the genus-Cephalosporium, than from among other mutants induced by other conventional mutation treatment, and that some of the polyploid molds are able to accumulate CPC extracellularly in unprecedentedly high concentrations.

Though attempts have heretofore been made to improve the fermentation yields of contemplated products by microbial polyploid strains, none of the reported cases have been industrially successful, either because of the failure to achieve satisfactory results or because of the difficulty with which these strains lend themselves to genetic fixation of characters. Of course, there is not a single reported case of attempt in connection with CPC fermentation.

This invention is directed to a method for the production of CPC comprising cultivating the polyploid microorganisms induced from parent microorganisms of the genus Cephalosporium having CPC producing ability in a culture medium to let the microorganism accumulate CPC extracellularly.

As the parent strains of microorganisms that are employed in this invention, use can be made of any suitable CPC producer of the genus Cephalosporium, for example, Cephalosporium acremonium or Cephalo- 5 ATCC-14553, etc., and various mutants induced from these strains.

The FERM-P and ATCC numbers indicate the accession number of the organisms at the Research lnstitute of Fermentation of the Agency of Industrial Science and Technology, Chiba, Japan and at American Type Culture Collection, Rockville, Md., U.S.A., respectively.

The following two procedures may be cited as principal procedures for inducing desired polyploid microorganisms from parent microorganisms.

One of them is the procedure comprising polyploidizing microbial cells with a chemical agent. As examples of said chemical agent, there may be mentioned camphor, acenaphthene, colchicine, etc. While the conditions of treatment vary with the particular mold, type of chemical agent and other factors, the following procedure generally can be followed with success. Thus, in the case of camphor or acenaphthene, for instance, 1 microbial cells are exposed to a vapor'of the chemical for a few to tens of hours, after which they are removed from the ambient vapor and further cultivated. In another scheme, the cells are cultivated from the beginning on an agar plate containing the chemical. The grown cells taken from the resulting colonies are examined, e.g., under a microscope. The colonies made up of giant cells are selectively taken.

By either of the above-described procedures there can be obtained the desired polyploid mold. For example, a polyploid mold can be obtained by the method described in Journal of General and Applied Microbiology 2, 345(1956). These procedures cannot only be followed in repetition but can be followed in combination with other methods of breeding and selecting some specific microbial strains and, in such instances, im-

proved results are sometimes obtained.

The polyploid strains thus obtained can be easily isolated, if desired, by a conventional pure-isolation method, for example by micromanipulation.

To illustrate, Cephalosporium acremonium ATCC- 14553 as a parent strain was treated with camphor and the CPC-producing abilities of the resulting polyploid strains were investigated. The investigation resulted in the finding of the number of strains which can accumulate CPC in the medium in the increased yields, for

example, more than 7,000 ug/ml, with Cephalosporium acremonium 2M-16 (FERM-P No.2283, ATCC-20425, [PO-9999), in particular, giving an unprecedentedly high yield of CPC. The taxonomic characters of this strain were found to be essentially identical with those of Cephalosporium sp. ATCC-14553 (Japanese patent publication No.4437/ 1965) except the differences noted in Table. l.

The [F0 number indicates the accession numbers of the organisms at the lnstitute for Fermentation, Osaka,

sporium polyaleurum. Japan.

Table 1 Size of Number of DNA content Amount of DNA arthrospore nuclei per per per nucleus Major Minor arthrospore arthrospore Strain diameter diameter (A) (B) (BIA) (n) (u) (us) (#2) Cephalosporium Tablel-continued Size of Number of DNA content Amount of DNA arthrospore nuclei per per per nucleus Major Minor arthrospore arthrospore Strain diameter diameter (A) (B) (B/A) (F) (n) (us) (as) acremonium 7.2 3.6 1.0 0.30 X 0.30 X 10" ATCC-14553 I Cephalosporium acremonium 11.2 4.7 3.2 1.98 X 10 0.62 X 10 2M-l6 (Note: All the values given in the above table are the 1 results on analysis of the arthrospores obtained after 2 many superior CFC-producing strains which can accumulate CPC extracellularly in the concentration of, for

weeks incubation at 28C on an agar medium comexample, more than 7,000 ug/ml, with Cephalosporium posed of 0.25 yeast extract, 0.5 malt extract, 0.1 acremonium LA-101 (FERM-P No.2284, ATCC- casein hydrolyzate (Casamino acid), 2.0 sucrose, 1.5 20426, lFO-3000l in particular, giving an unpreceglycerin and 2.0 agar. The dimentions of the ardentedly high yield of CPC. throspore and the number of nuclei are the mean of The taxonomic characters of this strain were found to results for approximately 100 arthrospores'The mean be essentially identical with the characters of Cephalodimensions of conidia are 3.5 p X 7.1 p. in the case of sporium sp. ATCC-14553 except the differences noted ATCC-l4553 and 4.3 a X 8.6 u in the case of 2M-l6). in Table 2.

Table 2 Size of conidium Number of DNA content DNA content Strain Major Minor nuclei per per conidium per nucleus diameter diameter conidium (B/A) (u) 1 (IL) (us) Cephalosporium acremonium 10.5 2.5 1 0.691 x 10- 0.691 x 10' ATCC-l4553 Cephalosporium acremonium 7.5 2.3 1 0.670 X 10 0.670 X 10" LM-5 Cephalosporium acremonium 7.3 1 2.7 1 0.710 X 10" 0.710 X 10" AP-78 Cephalosporium acremonium 7.9 3.3 1 1.380 X 10" 1.380 X 10' LA-lOl The second genetic method typically comprises the procedure described in Journal of General and Applied (Note: Each of the values given above is the result of Microbiology 2, 401 1956). Thus, the two strains to be analysis. of the conidia harvested from a slant culture crossed are differently marked beforehand, e.g., with grown by the procedure set forth in the footnote to different amino acid or vitamin requirements. They are Table 1. The dimensions of conidia and the number of crossed and grown on a minimal medium (e.g., a menuclei are the mean values for about 100 spores.) dium made up of 3.0 sucrose, 0.3 NH N0 ,0.l To cultivate these polyploid strains, the known meth- K HPO. 0.05 MgSO 7H O, a trace of FeSO .7H O ods for culture of other CPC producers of the genus and 1.5 agar) in the conventional manner. The 00- Cephalosporium or those of the genus Emericellopsis nidia of grown colonies are collected and plated onto a can be employed to advantage. minimal medium such as the one indicated above and As the carbon sources that can be. employed, there the colonies formed are investigated. Most of the may be mentioned, for example, glucose, sucrose, growth consists of polyploid strains. starch, soluble starch, waste molasses, n-paraffins, If necessary, these methods cannot only be practiced acetic acid, methanol, ethanol, etc. As the organic in repetition but can also be carried out in combination nitrogen sources, mention may be made of urea, meat with other selective .breeding procedures, and imextract, peptone, soybean meal, cotton seed cake, peaproved results can be obtained thereby. The resultant nut cake, dried yeast, corn steep liquor, etc., while polyploid strain can be easily isolated, if desired, by a ammonium chloride, ammonium sulfate, ammonium known pure-isolation method such as micromanipulanitrate, ammonium phosphate, potassium nitrate, etc. tion. may be mentioned as the useful inorganic sources of Using Cephalosporium acremonium LM-S, which is a nitrogen. If necessary, there may be added such metal methionine-leucine-requiring mutant, and Cephalosposalts as the 80,, Cl, N0 CO P0 and other salts of rium acremonium AP-78, an arginine-phenylalanine- Na, K, Ca, Mg, Mn, Zn, Fe, Cu, etc. requiring mutant, both induced from Cephalosporium Furthermore, there may also be added, as required. acremonium ATCC-14553 as the parent strain, a numamino acids (e.g., methionine, cysteine, serine), thiosulfates, fatty acid esters, oils, e.g., lard oil, olive oil,

ber of polyploid strains were obtained by the procedures hereinbefore described.

An investigation of the abilities of these polyploid' strains to accumulate CPC resulted in the finding of etc., and other agents which assist in the production of CPC, whereby the potency of CPC can be further increased.

As to the conditions of cultivation, aerated submerged culture is generally advantageous. The temperature is desirably within the range of 18 to 35C. Satisfactory results are obtained when the pH is maintained within the range of about 2 to and, preferably, between 4 and 9. A cultivation time of 3 to days is sufficient. Most of the CPC elaborated occurs in filtered broth and, therefore, to recover the antibiotic, it is advantageous to remove the cells from the broth by centrifugation or filtration and, then, purify CPC from the supernatant or filtrate. To isolate the CPC, the procedures generally used for the fractional recovery of CPC can be followed with success. For example, the desired result can be obtained with advantage by utilizing an ion exchange resin, activated carbon, nonionic copolymer resin, gel filtration medium, etc. in a suitable combination. The following examples are merely illustrative of this invention and should by no means be construed as limiting the scope of the invention.

EXAMPLE 1 A Sakaguchi flask of 2-liter capacity is filled with 500 ml of an inoculum medium made up of 3.0 of sucrose, 1.5 of meat extract, 0.5 of corn steep liquor and 0.15 of CaCO and, after sterilization, inoculated with Cephalosporium acremonium 2M-16. The flask is incubated on a reciprocating shaker at 28C for 3 days. Meanwhile, a 50-1iter fermentation tank of stainless steel is charged with 30 liters of a medium made up of 6 of sucrose, 5 of glucose, 3 of peanut cake, 3 of soybean meal, 1.0 of DL-methionine and 0.15 of CaCO and the medium is sterilized and cooled in the routine manner. The medium is then aseptically inoculated with the above seed culture and incubated at 28C under sparging and agitation (30 l. per min. aeration, 250 rpm.)

After 190 hours of cultivation, the culture broth is withdrawn and filtered free of solids. The resultant filtrate (25 l.) is assayed for CPC. The potency found is 8,200 ag/ml. The fractional isolation of CPC is carried out in the following manner. The filtrate (25 l.) is run down a column of activated carbon (20 1.), whereby the CPC is adsorbed on the carbon. The carbon is first washed well with water and, then, eluted with 5 aqueous butanol containing 0.01 N NaOH, whereupon 31 l. of CPC fractions are obtained. These fractions are pooled, concentrated and neutralized to pH 7.0 with NaOH. Finally, ethanol is added to the concentrate to a concentration of 50 (V/V).

The procedure gives 143 g. crude crystals of CPC sodium dihydrate.

The potency of CPC is measured by an enzymatic assay method using the cephalosporinase of Aerobacter cloaceae [Biochemical Journal, 116, 385 (1970)].

EXAMPLE 2 The same cultivation as in Example 1 is carried out, except that the strain used is Cephalosporium aoremonium LA-101. The potency of CPC in the filtered broth is 7,900 pg/ml.

What is claimed is:

1. A process for producing cephalosporin C which comprises cultivating Cephalosporium acremom'um ATCC 20425 or Cephalosporium acremonium ATCC 20426 in a culture medium containing an assimilable carbon source and a digestible nitrogen source, to accumulate cephalosporin C in the culture medium, and recovering the accumulated cephalosporin C from the culture medium.

2. A process according to claim 1, wherein the microorganism is Cephalosporium acremonium ATCC-20425 3. A process according to claim 1, wherein the microorganism is Cephalosporium acremonium ATCC- 20426. 

1. A PROCESS FOR PRODUCING CEPHALOSPORIN C WHICH COMPRISES CULTIVATING CEPHALOSPORIUM ACREMONIUM ATCC 20425 OR CEPHALOSPORIUM ACREMONIUM ATCC 20426 IN A SULTURE MEDIUM CONTAINING AN ASSIMILABLE CARBON SOURCE AND A DIGESTIBLE NITROGEN SOURCE, TO ACCUMULATE CEPHALOSPORIN C IN THE CULTURE MEDIUM, AND RECOVERING THE ACCUMULATED CEPHALOSPORIN C FROM THE CULTURE MEDIUM.
 2. A process according to claim 1, wherein the microorganism is Cephalosporium acremonium ATCC-20425
 3. A process according to claim 1, wherein the microorganism is Cephalosporium acremonium ATCC-20426. 